The present invention relates to a novel transcriptional regulator containing a bromodomain and a gene encoding it.
The bromodomain is a characteristic motif of proteins found in transcriptional regulators. Proteins having a bromodomain usually contain one or two (Tamkun, J. W. et al., (1992), Nuc. Acids Res., 20:2603), but sometimes as many as five bromodomain motifs (Nicolas, R. H. and Goodwin, G. H. (1996), Gene, 175 (12):233-240). This motif is found in a wide variety of animals. For example, it is identified in yeast (Winston, F. et al., (1987), Genetics, 115:649-656; Laurent, B. C. et al., (1991), Proc. Natl. Acad. Sci. USA, 88:2687-2691), in Drosophila (Digan, M. E. et al., (1986), Dev. Biol., 114:161-169; Tamkun, J. W. et al., (1992), Cell, 68:561-572), and in the genes for transcriptional regulators in mammals (Denis, G. V. and Green, M. R. (1996), Genes and Devel., 10:261-271; Yang, X. J. et al., (1996), Nature, 382:319-324).
All transcriptional regulators containing a bromodomain serve to control signal-dependent transcription in actively proliferating cells (Tamkun, J. W. et al., (1992), Cell, 68:561-572; Haynes, S. R. et al., (1992), Nuc. Acids Res., 20:2603). Due to this feature of these transcriptional regulators, it is suggested that cancer may develop if the gene for the protein containing a bromodomain is not normally controlled. In fact, several studies have shown that human transcriptional regulators with a bromodomain RING3, p300/CBP, and PCAF may be involved in oncogenesis.
RING3 is a transcriptional regulator highly homologous with the fsh protein that regulates development of Drosophila (Haynes, S. R. et al., (1989), Dev. Biol., 134:246-257). RING3 is a nuclear serine/threonine kinase having autophosphorylating activity. This activity of RING3 correlates with a proliferating state in chronic or acute lymphocytic leukemia. For instance, when Denis and Green collected lymphocytes of peripheral blood from 10 patients suffering from leukemia, kinase activity associated with RING3 was identified in all of the 10 patients but not in normal controls (Denis, G. V. and Green, M. R. (1996), Genes and Develop., 10:261-271). Furthermore, this activity was not detected in the blood cells from patients whose leukemia had remitted by virtue of chemotherapy.
p300 and CBP (CREB binding protein) encode highly similar proteins and are thus often called p300/CBP. p300/CBP is a co-activatot for a transcriptional regulator CREB (cAMP responsive element binding protein) (Kwok, RPS et al., (1994), Nature, 370:223-226), and is considered as a key protein for growth regulation. Mutation in p300/CBP has been found in familial or sporadic cancers. Germline mutation of CBP results in Rubinstein-Taybi syndrome, which causes patients to develop various malignant tumors (Petrij, F. et al., (1995), Nature, 376:348-51), while mutation in p300 is found in sporadic colorectal and gastric cancers (Muraoka, M. et al., (1996), Oncogene, 12:1565-1569). Furthermore, CBP is fused with MOZ (Monocytic leukemia Zinc finger protein) in a t (8; 16) (p11; p13) translocation found in a certain kinds of acute myelocytic leukemia. The fusion protein has histone-acetyltransferase domains derived from both genes (Bannister, A. J. and Kouzarides, T. (1996), Nature, 384:641-643; Orgyzco, V. V. et al., (1996), Cell, 87:953-959; Brownwell, J. E. and Allis, C. D. (1996), Curr. Opin. Genet. Devel., 6:176-184). Since acetylated histone is known to be associated with transcriptionally active chromatin, the fusion protein may be involved in leukemogenesis by way of aberrant histone acetylation (Brownwell, J. E. and Allis, C. D. (1996), Curr. Opin. Genet. Devel., 6:176-184).
p300/CBP is also considered to be associated with cancer since it interacts with known oncogene products. For example, p300/CBP binds to E1A protein (Arany, Z. et al., (1995), Nature, 374:81-84), one of the early genes of adenovirus. p300 is also a co-activator for transcription factors, c-Myb (Dai, P. et al., (1996), Genes Dev., 10:528-540) and c-Fos (Bannister, A. J. and Kouzarides, T. (1996), Nature, 384:641-643).
PCAF, is considered to inhibit the interaction of E1A with p300/CBP by competing with E1A for binding to p300/CBP (Yang, X. J. et al., (1996), Nature, 382:319-324). PCAF also has histone-acetyltransferase activity.
Thus, it is thought that transcriptional regulators containing a bromodomain areinvolved in regulation ofcell growth, and that their aberrant regulation may be closely related to various diseases, particularly to cancer. Transcriptional regulators containing a bromodomain have thus recently received much attention as novel targets for specifically treating cancer.
The objective of the present invention is to provide a novel transcriptional regulator containing a bromodomain and a gene encoding it, and a method of screening for a candidate compound as a medicament by using them.
As a result of research to achieve the above objective, the inventors successfully isolated several genes, each of which encodes a novel transcriptional regulator containing a bromodomain. The genes were isolated from a human testis cDNA library using primers designed based on EST sequences which had been identified using known bromodomain sequences as probes. In addition, the inventors have found that the structures of the isolated genes resemble one another, thus they constitute a family. The inventors have also found that the isolated genes or proteins encoded by them can be used to screen the candidate compounds for a medicament that controls the activity of the proteins or other factors interacting therewith.
Thus, the present invention relates to novel transcriptional regulators each having a bromodomain and the genes encoding them, and to a method of screening for a candidate compound as a medicament using said proteins or genes, and more specifically relates to:
(1) a transcriptional regulator having a bromodomain, which comprises the amino acid sequence shown in SEQ ID NO:1, 13, 21, 27, or 29, or said sequence wherein one or more amino acids are substituted, deleted, or added;
(2) a transcriptional regulator having a bromodomain, which is encoded by DNA hybridizing with DNA comprising the nucleotide sequence shown in SEQ ID NO:2, 14, 22, 28 or 30;
(3) DNA coding for the transcriptional regulator according to (1) or (2);
(4) a vector comprising the DNA according to (3);
(5) a transformant expressibly retaining the DNA according to (3);
(6) a method for producing the transcriptional regulator according to (1) or (2), which comprises culturing the transformant according to (5);
(7) an antibody binding to the transcriptional regulator according to (1) or (2);
(8) a method of screening a compound having binding activity to the transcriptional regulator according to (1) or (2), wherein the method comprises contacting a sample with the transcriptional regulator according to (1) or (2) and selecting a compound having binding activity to the transcriptional regulator according to (1) or (2);
(9) a compound having binding activity to the transcriptional regulator according to (1) or (2), which can be isolated according to the method of (8);
(10) the compound according to (9), which is naturally occurring; and
(11) DNA specifically hybridizing with DNA comprising the nucleotide sequence shown in SEQ ID NO:2, 14, 22, 28, or 30 and having at least 15 nucleotides.
Here, the term xe2x80x9ctranscriptional regulator(s)xe2x80x9d means protein(s) that control gene expression, and xe2x80x9cbromodomainxe2x80x9d means an amino acid motif conserved among the transcriptional regulators associated with signal-dependent transcription, wherein said motif is involved in protein-protein interaction.
The present invention relates to novel transcriptional regulators having a bromodomain (BAZ family). The nucleotide sequences of cDNA isolated by the inventors, which belong to BAZ family, are shown in SEQ ID NO:2 (BAZ(BAZ1xcex1)), SEQ ID NO:14 (BAZ2xcex1), SEQ ID NO:22 (BAZ2xcex2), and SEQ ID NO:28 and 30 (BAZ1xcex2). The amino acid sequences of proteins encoded by the cDNA are also shown in SEQ ID NO:1 (BAZ(BAZ1xcex1)), SEQ ID NO:13 (BAZ2xcex1), SEQ ID NO:21 (BAZ2xcex2), and SEQ ID NO:27 and 29 (BAZ1xcex2).
The bromodomain is characteristic of a structural region that is conserved among a group of transcriptional regulators involved in signal-dependent transcription (Tamkun, J. W. et al., (1992), Cell, 68:561-572; Haynes, S. R. et al., (1992), Nuc. Acids Res., 20:2603), and it has been reported that the six mammalian genes, i.e., RING3, p300/CBP, PCAF, BRG1, HRX/ALL-1, and TIF1, which encode transcriptional regulators having a bromodomain, are associated with cancer. That the transcriptional regulators having a bromodomain are commonly associated with cancer suggests that the genes isolated by the inventors are also associated with cancer. Other than a bromodomain motif, the proteins encoded by the genes isolated by the inventors share the characteristic motifs of (1) C4HC3 zinc-finger, which is found in the proteins expressed in a wide range of organisms from yeast to human and is believed to be involved in a protein-protein interaction or nonspecific binding to DNA; (2) leucine zipper, which is present in many transcriptional regulators and is known to contribute to form a dimer with the protein itself or other proteins (Busch, S. J. and Sassone-Corsi, P (1990), Trends in Genetics, 6:36-40); (3) LXXLL (SEQ ID NO:37) motif, a motif commonly found among many transcriptional co-activators, which is shown to be required for mediation of transcription induced by a nuclear receptor (Torchia, J. et al., (1997), Nature, 387:677-684; Heery, D. M. et al., (1997), Nature, 387:733-736); and (4) nuclear transport signal, which confers the transporting activity into the nucleus on the proteins synthesized in the cytoplasm.
The combination of a bromodomain and C4HC3 zinc finger is known to be associated with several breakpoint genes of leukemia (Tkachuk, D. C. et al., (1992), Cell, 71:691-700; Gu, Y. et al., (1992), Cell, 71:701-798; Miki, T. et al., (1991), Proc. Nat. Acad. Sci., 88:5167-5171; Le Douarin B. et al., (1995), EMBO J., 14:2020-2033; Borrow, J. et al., (1996), Nature Genet., 14:33-41). Accordingly, the genes isolated by the inventors are important candidates for breakpoint genes of cancers.
The transcriptional regulators of the present invention can be prepared as recombinant proteins generated using a recombinant gene technique, or as naturally occurring proteins, according to a method known to one skilled in the art. The recombinant proteins can be prepared using a method such as incorporating DNA encoding a transcriptional regulator of the present invention (e.g., DNA having the nucleotide sequence shown in SEQ ID NO:2, 14, 22, 28, or 30) into a suitable expression vector, which is then introduced into host cells, and purifying the protein obtained from the transformant. The naturally occurring proteins can be prepared using a method such as preparing a column which utilizes an antibody obtained from a small animal immunized with the recombinant protein prepared as above, and subjecting the extract from a tissue or cells in which a transcriptional regulator of the present invention is overexpressed (e.g., testis and cancer cells) to affinity chromatography using said column.
The present invention also relates to transcriptional regulators functionally equivalent to the transcriptional regulators of the present invention having the amino acid sequence shown in SEQ ID NO:1, 13, 21, 27, or 29. A method of introducing mutation into amino acids of a protein to isolate a protein functionally equivalent to a particular protein is well known to one skilled in the art. Thus, it is well within the art of an ordinarily skilled person to isolate a transcriptional regulator functionally equivalent to the transcriptional regulators of the present invention having the amino acid sequence shown in SEQ ID NO:1, 13, 21, 27, or 29 by appropriately modifying, for example, substituting amino acids without affecting the function of the transcriptional regulator. Mutation in an amino acid of a protein can also occur spontaneously. The transcriptional regulators of the present invention include those having a bromodomain and the amino acid sequence of SEQ ID NO:1, 13, 21, 27, or 29 wherein one or more amino acids are substituted, deleted, or added. Examples of known methods for introducing amino acid mutation into the protein are a site-directed mutagenesis system using PCR (GIBCO-BRL, Gaithersburg, Md.) and a site-directed mutagenesis using oligonucleotides (Kramer, W. and Fritz, H. J. (1987), Methods in Enzymol., 154:350-367). The number of mutagenized amino acids is usually 50 amino acids or less, preferably 30 amino acids or less, more preferably 10 amino acids or less, and most preferably three amino acids or less.
As another method of isolating a functionally equivalent protein utilizing a hybridization technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to one skilled in the art. Based on the DNA sequence encoding the transcriptional regulator of the present invention shown in SEQ ID NO:2, 14, 22, 28, or 30, or the fragment thereof, a person with ordinary skill in the art can isolate DNA highly homologous to said DNA sequences using a hybridization technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) to obtain a transcriptional regulator functionally equivalent to the transcriptional regulators. The transcriptional regulators of the present invention include those encoded by DNA that hybridizes with DNA comprising the DNA sequence shown in SEQ ID NO:2, 14, 22, 28, or 30, and which contains a bromodomain. The hybridization and washing conditions for isolating DNA encoding a functionally equivalent protein are defined as low stringency: 42xc2x0 C., 2xc3x97SSC, 0.1% SDS; moderate stringency: 50xc2x0 C., 2xc3x97SSC, 0.1% SDS; and high stringency: 65xc2x0 C., 2xc3x97SSC, 0.1% SDS. The transcriptional regulators obtained by the hybridization technique may have amino acid homology of preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, or most preferably 95% or more, with the transcriptional regulators having the amino acid sequence shown in SEQ ID NO:1, 13, 21, 27, or 29. In particular, high homology in the bromodomain sequence is considered significant for the function associated with cancer. Functionally equivalent transcriptional regulators to be isolated may contain, other than a bromodomain, a sequence involved in interaction with another protein (e.g., leucine-zipper or LXXLL (SEQ ID NO:37) motif), a sequence involved in binding to DNA (e.g. zinc finger), or a nuclear transport signal. The presence of the bromodomain in the protein can be identified by searching the bromodomain motif PROSITE database on DNASIS (HITACHI Software engineering).
The present invention also relates to DNA that codes for a transcriptional regulator of the present invention. The DNA of the present invention includes cDNA, genomic DNA, and chemically synthesized DNA, but is not limited thereto as long as it codes for a transcriptional regulator of the present invention. cDNA can be prepared, for example, by designing a primer based on the nucleic acid sequence shown in SEQ ID NO:2, 14, 22, 28, or 30 and performing plaque PCR (see Affara, N. A. et al., (1994), Genomics, 22:205-210). The genomic DNA can be prepared according to a standard technique using, for example, Qiagen genomic DNA kits (Qiagen, Hilden, Germany). The DNA sequence thus obtained can be determined according to a standard technique using a commercially available dye terminator sequencing kit (Applied Biosystems) and the like. In addition to applying to the production of recombinant proteins as described below, the DNA of the present invention may be applied to gene therapy and the like.
The present invention also relates to a vector into which the DNA of the present invention is inserted. There is no particular limitations to the vector into which the DNA of the present invention is inserted, and various types of vectors, e.g. for expressing the transcriptional regulators of the present invention in vivo and for preparing recombinant proteins, may be used for each purpose. Vectors used for expressing the transcriptional regulators of the present invention in vivo (in particular, for gene therapy) include the adenovirus vector pAdexLcw and the retrovirus vector pZIPneo. A LacSwitch II expression system (Stratagene; La Jolla, Calif.) is advantageous when mammalian cells, such as CHO, COS, and NIH3T3 cells, are used. An expression vector is particularly useful for producing a transcriptional regulator of the present invention. Although there is no particular limitation to the expression vectors, the following vectors are preferred: pREP4 (Qiagen, Hilden, Germany) when E. coli is used; SP-Q01 (Stratagene, La Jolla, Calif.) when yeast is used; and BAC-to-BAC baculovirus expression system (GIBCO-BRL, Gaithersburg, Md.) when insect cells are used. The DNA of the present invention can be inserted into vectors using a standard method.
The present invention also relates to a transformant expressibly retaining the DNA of the present invention. The transformants of the present invention include one harboring the above-described vector into which the DNA of the present invention is inserted and one having the DNA of the present invention integrated into its genome. The DNA of the present invention can be retained in the transformant in any form as long as the transformant expressibly retains the DNA of the present invention.. There is no limitation to host cells into which a vector of the present invention is introduced. If the cells are used to express a transcriptional regulator of the present invention in vivo, desired cells may be used as target cells. Cells such as E. coli, yeast cells, animal cells, and insect cells can be used for producing the transcriptional regulators of the present invention. The vector can be introduced into the cells by methods such as electroporation and heat shock. Recombinant proteins can be isolated and purified from the transformants generated for producing the said proteins according to a standard method.
The present invention also relates to antibodies that bind to the transcriptional regulators of the present invention. The antibodies of the present invention include, but are not limited to, polyclonal and monoclonal antibodies. Also included are antisera obtained by immunizing an animal such as a rabbit with a transcriptional regulator of the present invention, any class of polyclonal or monoclonal antibodies, humanized antibodies generated by gene recombination, and human antibodies. The antibodies of the present invention can be prepared according to the following method. For polyclonal antibodies, antisera can be obtained by immunizing a small animal, such as a rabbit, with a transcriptional regulator of the present invention, then recovering the fractions that only recognize the transcriptional regulator of the present invention through an affinity column coupled with the transcriptional regulator of the present invention. Immunoglobulin G or M can be prepared by purifying the fractions through a Protein A or G column. For monoclonal antibodies, a small animal, such as a mouse, is immunized with a transcriptional regulator of the invention, the spleen is removed from the mouse and homogenized into cells, the cells are fused with myeloma cells from a mouse using a reagent such as polyethylene glycol, and clones that produce antibodies against the transcriptional regulator of the invention are selected from the resulting fused cells (hybridoma). The hybridoma obtained is then transplanted into the abdominal cavity of a mouse, and the ascites are recovered from the mouse. The obtained monoclonal antibodies can then be prepared by purifying, for example, by ammonium sulfate precipitation through a Protein A or G column, by DEAE ion exchanging chromatography, or through an affinity column coupled with the transcriptional regulator of the invention. Besides being used to purify or detect the transcriptional regulators of the present invention, the antiobodies of the present invention can be applied to antibody therapy.
The present invention also relates to a screening method for a compound that binds to transcriptional regulators of the present invention. The screening method of the present invention includes steps of contacting a transcriptional regulator of the present invention with a test sample and selecting a compound that has binding activity for the transcriptional regulator of the present invention. Test samples used for the screening include, but are not limited to, a cell extract, a supernatant of the cell culture, a library of synthetic low molecular weight compounds, a purified protein, an expression product of a gene library, and a library of synthetic peptides. Methods well known to one skilled in the art for isolating a compound binding to a transcriptional regulator of the present invention using the regulator are as follows. A protein that binds to a transcriptional regulator of the present invention can be screened by West-western blotting comprising steps of generating a cDNA library from the cells expected to express the protein that binds to a transcriptional regulator of the present invention (e.g., testis tissue cell and tumor cell lines HL-60, HeLa S3, Raji, and SW480) using a phage vector (xcexgt11, ZAP, etc.), allowing the cDNA library to express on the LB-agarose plate, fixing the expressed proteins on a filter, reacting them with the transcriptional regulator of the present invention purified as a biotin-labeled protein or a fusion protein with GST protein, and detecting plaques expressing the protein bound to the regulator on the filter with streptavidin or anti-GST antibody (Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fisher, R., Drepps, A., Ullrich, A. and Schlessinger, J. (1991), Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases, Cell, 65:83-90). Alternatively, the method comprises expressing in yeast cells a transcriptional regulator of the present invention which is fused with SFR or GAL4 binding region, constructing a cDNA library in which proteins are expressed in a fusion protein with the transcription activation site of VP16 or GAL4 from the cells expected to express a protein that binds to the transcriptional regulator of the present invention, introducing the cDNA library into the above-described yeast cells, isolating the cDNA derived from the library from the detected positive clones, and introducing and expressing it in E. coli. (If a protein that binds to the transcriptional regulator of the present invention is expressed, a reporter gene is activated by the binding of the two proteins. The positive clones can then be identified.) This method can be performed using Two-hybrid system (MATCHMAKER Two-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit, MATCHMAKER One-Hybrid System (all from Clontech); HybriZAP Two-Hybrid Vector System (Stratagene) or in accordance with Dalton, S. and Treisman R. (1992), Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element, Cell, 68:597-612). Another method is to apply a culture supernatant or a cell extract from the cells suspected to express a protein which binds to the transcriptional regulator of the present invention onto an affinity column coupled with the transcriptional regulator of the present invention, and purify the protein specifically bound to the column.
Also well known to one skilled in the art are a method of screening molecules that bind to a transcriptional regulator of the present invention by reacting the immobilized transcriptional regulator of present invention with a synthetic compound, natural substance bank, or a random phage peptide display library, and a method of screening low molecular weight compounds, proteins (or their genes), or peptides which bind to a transcriptional regulator of the present invention by utilizing the high-throughput technique of combinatorial chemistry (Wrighton, N. C., Farrell, F. X., Chang, R., Kashuyap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., Dower, W. J., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (UNITED STATES) Jul. 26, 1996, 273:458-464; Verdine, G. L., The combinatorial chemistry of nature, Nature (ENGLAND), Nov. 7, 1996, 384:11-13; Hogan, J. C. Jr., Directed combinatorial chemistry, Nature (ENGLAND), Nov. 7, 1996, 384:17-19). The compounds thus isolated, which bind to a transcriptional regulator of the present invention, may be used to treat cancer or other proliferative diseases. When the compounds isolated by the screening method of the present invention are used as pharmaceuticals, they can be formulated by a known pharmacological process. For example, they can be administered to a patient with pharmaceutically acceptable carriers and vehicles (e.g., physiological saline, vegetable oil, a dispersant, a surfactant, and a stabilizer). The compounds may be percutaneously, intranasally, transbronchially, intramuscularly, intravenously, or orally administered, depending on their properties.
The present invention also relates to DNA specifically hybridizing with DNA coding a protein of the present invention and having at least 15 nucleotides. As used herein, xe2x80x9cspecifically hybridizingxe2x80x9d means that no cross-hybridization occurs between DNA encoding other proteins under conditions of moderate stringency. Such DNA may be used as a probe for detecting and isolating the DNA encoding the protein of the present invention, and as a primer for amplifying the DNA encoding the protein of the present invention.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, and (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide was associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
As used herein, xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. These programs are available at the web site of the National Center for Biotechnology Information.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.